Structural basis of eIF4E recognition of synthetic mRNA 5′ cap analogues links cap architecture to affinity and biological activity
Abstract
Eukaryotic mRNAs carry a 5′-terminal 7-methylguanosine (m7G) cap that is recognized by multiple cellular proteins and is essential for mRNA self-discrimination, stability, and translation. The eukaryotic translation initiation factor 4E (eIF4E) binds the 5′ cap and promotes recruitment of mRNAs to the ribosomal translation initiation complex, a critical step in cap-dependent translation. Beyond naturally occurring cap modifications that modulate mRNA interactions and fate, non-natural 5′ cap modifications can be used to finely tune diverse aspects of mRNA biology. This has gained particular relevance with the emergence of in vitro transcribed mRNAs as a powerful therapeutic modality for protein replacement therapies and vaccines. Here, we report binding data for a series of synthetic 5′ cap analogues interacting with eIF4E, together with X-ray crystal structures of eIF4E-cap complexes, providing detailed insights into the structural determinants of cap recognition by eIF4E. Furthermore, analysis of gene expression from mRNAs carrying different synthetic cap analogues in human cell lines and mice revealed a relationship between cap architecture and protein expression. Taken together, our structural, biochemical, and functional data establish structure-activity relationships for synthetic cap analogues and identify structural features associated with enhanced translational performance. GRAPHICAL ABSTRACT RNA icon by Servier https://smart.servier.com/ is licensed under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/ NIAID Visual & Medical Arts. (10/7/2024). Lab Mouse. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/281 NIAID Visual & Medical Arts. (10/7/2024). Syringe. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/505 NIAID Visual & Medical Arts. (10/7/2024). HEK 293 Cells. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/198 NIAID Visual & Medical Arts. (10/7/2024). Cap Binding Protein. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/66 RNA CC-BY 3.0 unported